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Axenfeld-Rieger Syndrome Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
Order Kits
TEST METHODS

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1995 ASPH 81479 Add to Order
B3GLCT 81479
COL4A1 81408
CYP1B1 81404
FOXC1 81479
FOXE3 81479
PAX6 81479
PITX2 81479
SH3PXD2B 81479
Full Panel Price* $680.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1995 Genes x (9) $680.00 81404, 81408, 81479(x7) Add to Order
Pricing Comment

We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.

Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

Approximately 25%-60% of Axenfeld-Rieger syndrome cases are due to FOXC1 or PITX2 pathogenic variants (Tümer and Bach-Holm. 2009. PubMed ID: 19513095; Reis et al. 2012. PubMed ID: 22569110; Alward. 2000. PubMed ID: 11004268). In one study, two out of 30 Axenfeld-Rieger syndrome patients (6.7%) carried SH3PXD2B sequence variants (Mao et al. 2012. PubMed ID: 2250910). Due to the phenotypic and genotypic heterogeneity, clinical sensitivity of the other genes in this panel is difficult to predict.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 B3GLCT$990.00 81479 Add to Order
CYP1B1$990.00 81479
FOXC1$990.00 81479
FOXE3$990.00 81479
PAX6$990.00 81479
PITX2$990.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (6) $1290.00 81479(x6) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$990

2-5

$1190

6-10

$1290

11-100

$1490

Over 100

Call for quote
Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Large deletions/duplications or complex genomic rearrangements have been reported in FOXC1, PAX6, CYP1B1, B3GLCT and PITX2 (Human Gene Mutation Database).

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Clinical Features

Axenfeld-Rieger syndrome (ARS) is a rare, highly penetrant disorder characterized by varying degrees of eye anterior segment anomalies with systemic malformations such as dental hypoplasia and a protuberant umbilicus (Hjalt and Semina. 2005. PubMed ID: 16274491; Berry et al. 2006. PubMed ID: 16449236; Waldron et al. 2010. PubMed ID: 20831741; Tümer and Bach-Holm. 2009. PubMed ID: 19513095; Chang et al. 2012. PubMed ID: 22199394). Dental abnormalities in this syndrome help in diagnosis and to distinguish ARS from other eye anterior segment abnormalities. Early diagnosis of ARS from its dento-facial and systemic features is essential in treating or preventing the most serious consequence of ARS (O'Dwyer and Jones. 2005. PubMed ID: 16238657). The major clinical concern is high risk of developing open-angle glaucoma, which represents the main challenge in terms of treatment. Approximately 50% of ARS affected patients develop glaucoma (Shields et al. 1985. PubMed ID: 389274; Alward. 2000. PubMed ID: 11004268; Hjalt and Semina. 2005. PubMed ID: 16274491; Chang et al. 2012. PubMed ID: 22199394). ARS affected patients also need surveillance and management of sensorineural hearing loss, and cardiac, endocrinological, craniofacial and orthopaedic defects (Chang et al. 2012. PubMed ID: 22199394). Rieger syndrome (RIEG) and Peters anomaly both are in the anterior chamber cleavage group of anomalies and are considered to be variations of a single developmental disorder (the ARS group) (Reese and Ellsworth. 1966. PubMed ID: 5948260). RIEG is characterized by malformations of the eyes, teeth, and umbilicus; whereas Peters anomaly displays only ocular features (Amendt et al. 2000. PubMed ID: 11092457; Doward et al. 1999. PubMed ID: 10051017).

Genetics

Pathogenic variants in the FOXC1 or PITX2 genes cause autosomal dominant (AD) Axenfeld-Rieger syndrome (ARS). Linkage studies identified four chromosomal loci (4q25, 6p25, 13q14 and 16q24) that are associated with ARS and related or overlapping phenotypes. The genes that have been identified on chromosomes 4q25 and 6p25 are PITX2 (the pituitary homeobox 2 gene) and FOXC1 (the forkhead box C1 gene, also known as FKHL7) respectively (Alward. 2000. PubMed ID: 11004268; Lines et al. 2002. PubMed ID: 12015277; Tümer and Bach-Holm. 2009. PubMed ID: 19513095). PITX2 and FOXC1 are transcription factor genes which are expressed throughout eye ontogeny (Lines et al. 2002. PubMed ID: 12015277). The genes at 13q14 and 16q24 have not yet been discovered.

PITX2 encodes a Paired-like homeodomain transcription factor (PITX2), which plays a critical role in cell proliferation, differentiation, hematopoiesis and organogenesis (Huang et al. 2009). Also, PITX2 integrates retinoic acid and canonical Wnt signaling during eye anterior segment development (Gage et al. 2008. PubMed ID: 18367164; Gage and Zacharias. 2009. PubMed ID: 19623614 ). The product of the FOXC1 gene (FOXC1) is reported to maintain homeostasis in trabecular meshwork (TM) cells by regulating genes that play an important role in stress response (Ito et al. 2014. PubMed ID: 24556684; Paylakhi et al. 2013. PubMed ID: 23541832). TM helps in regulating intraocular pressure by acting as a drainage structure for aqueous humor (Tamm. 2009. PubMed ID: 19239914). Pathogenic mutations in FOXC1 significantly decrease the TM cell viability and subsequently contribute to the development of glaucoma.

A Genotype-Phenotype correlation study indicated that patients with PITX2 pathogenic variants have a more severe prognosis for glaucoma as compared to FOXC1 pathogenic variants. Within FOXC1 pathogenic variants, patients with FOXC1 duplications are at higher risk for the development of glaucoma, which emphasizes the importance of genetic testing. It’s been reported that PITX2 and FOXC1 interact with each other, which is essential for the regulation of common downstream target genes within specific cell lineages (Berry et al. 2006. PubMed ID: 16449236). Also, co-inheritance of PITX2 and FOXC1 pathogenic variants has been reported in a family which segregated with the disease and showed variable phenotypic expression. The most severely affected individual had pathogenic variants in both genes, whereas single heterozygous variants caused milder ARS phenotypes (Kelberman et al. 2011). So far, about 80 and 100 ARS causative variants have been identified in the PITX2 and FOXC1 genes, respectively (Human Gene Mutation Database).

Pathogenic variants in COL4A1 also cause AD ARS (Sibon et al. 2007. PubMed ID: 17696175). Pathogenic variants in SH3PXD2B cause autosomal recessive (AR) Frank-ter Haar syndrome. SH3PXD2B analysis in Axenfeld-Rieger syndrome patients identified several rare variants. However, the pathogenicity of these have not been well documented (Mao et al. 2012. PubMed ID: 22509100). Pathogenic variants in PAX6 and CYP1B1 have been shown to be associated with AD Peters anomaly (Hanson et al. 1994. PubMed ID: 8162071; Vincent et al. 2001. PubMed ID: 11403040). Pathogenic variants in ASPH cause AR Traboulsi syndrome or facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs disorder (Patel et al. 2014. PubMed ID: 24768550). Pathogenic variants in FOXE3 cause AR anterior segment dysgenesis 2, multiple subtypes including Peters anomaly (Doucette et al. 2011. PubMed ID: 21150893). Pathogenic variants in B3GLCT cause AR Peters-plus syndrome (Lesnik Oberstein et al. 2006. PubMed ID: 16909395).

A wide variety of causative variants (missense, nonsense, splicing, small deletions and insertions) have been reported in Axenfeld-Rieger syndrome and overlapping phenotypes -associated genes including large deletions/duplications and complex genomic rearrangements in a few genes (FOXC1, PAX6, CYP1B1, B3GLCT and PITX2) (Human Gene Mutation Database).

See individual gene test descriptions for more information on molecular biology of gene products.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

This panel typically provides >99.8% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing. A small region in exon 1 of FOXE3 is not covered for technical reasons.

Indications for Test

All patients with symptoms suggestive of Axenfeld–Rieger syndrome, Rieger syndrome and Peters anomaly are candidates.

Genes

Official Gene Symbol OMIM ID
ASPH 600582
B3GLCT 610308
COL4A1 120130
CYP1B1 601771
FOXC1 601090
FOXE3 601094
PAX6 607108
PITX2 601542
SH3PXD2B 613293
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Aniridia via PAX6 Gene Sequencing with CNV Detection
Anterior Segment Dysgenesis via FOXE3 Gene Sequencing with CNV Detection
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
FOXC1-Related Disorders via the FOXC1 Gene
Peters Plus Syndrome via B3GALTL/B3GLCT Gene Sequencing with CNV Detection
PITX2- Related Disorders via PITX2 Gene Sequencing with CNV Detection
Primary Congenital Glaucoma via the CYP1B1 Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Alward. 2000. PubMed ID: 11004268
  • Amendt et al. 2000. PubMed ID: 11092457
  • Berry et al. 2006. PubMed ID: 16449236
  • Chang et al. 2012. PubMed ID: 22199394
  • Doucette et al. 2011. PubMed ID: 21150893
  • Doward et al. 1999. PubMed ID: 10051017
  • Gage and Zacharias. 2009. PubMed ID: 19623614
  • Gage et al. 2008. PubMed ID: 18367164
  • Hanson et al. 1994. PubMed ID: 8162071
  • Hjalt and Semina. 2005. PubMed ID: 16274491
  • Huang et al. 2009. PubMed ID: 19174163
  • Human Gene Mutation Database (Bio-base).
  • Ito et al. 2014. PubMed ID: 24556684
  • Kelberman et al. 2011. PubMed ID: 21837767
  • Lesnik Oberstein et al. 2006. PubMed ID: 16909395
  • Lines et al. 2002. PubMed ID: 12015277
  • Mao et al. 2012. PubMed ID: 22509100
  • O'Dwyer and Jones. 2005. PubMed ID: 16238657
  • Patel et al. 2014. PubMed ID: 24768550
  • Paylakhi et al. 2013. PubMed ID: 23541832
  • Reese and Ellsworth. 1966. PubMed ID: 5948260
  • Reis et al. 2012. PubMed ID: 22569110
  • Shields et al. 1985. PubMed ID: 3892740
  • Sibon et al. 2007. PubMed ID: 17696175
  • Tamm. 2009. PubMed ID: 19239914
  • Tümer and Bach-Holm. 2009. PubMed ID: 19513095
  • Vincent et al. 2001. PubMed ID: 11403040
  • Waldron et al. 2010. PubMed ID: 20831741
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.
SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.
DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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